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Table 1 Ligands and binding protocols used for receptor autoradiography

From: Multireceptor fingerprints in progressive supranuclear palsy

Transmitter Receptor [3H]-Ligand Displacer Incubation buffer Preincubation Main incubation Final rinsea
Glutamate AMPA AMPA
[10 nM]
Quisqualate
[10 μM]
50 mM Tris-acetate (pH 7.2)
+100 mM KSCNb
3 × 10 minutes, 4 °C 45 minutes, 4 °C 1. 4 × 4 seconds
2. Acetone/glutaraldehyde (100 ml/2.5 ml), 2 × 2 seconds, 4 °C
  Kainate Kainate
[9.4 nM]
SYM 2081
[100 μM]
50 mM Tris-acetate (pH 7.1)
+ 10 mM Ca2+-acetate
3 × 10 minutes, 4 °C 45 minutes, 4 °C 1. 4 × 4 seconds, 4 °C
2. Acetone/glutaraldehyde (100 ml/2.5 ml), 2 × 2 seconds, 4 °C
  NMDA MK-801
[3.3 nM]
(+)MK-801
[100 μM]
50 mM Tris-acetate (pH 7.2)
+ 50 μM glutamate
+ 30 μM glycineb
+ 50 μM spermidineb
15 minutes, 4 °C 60 minutes, 22 °C 2 × 5 minutes
  mGlu2/3 LY341495
[1 nM]
l-Glutamate
[1 mM]
10 mM phosphate buffer (pH 7.6)
+ 100 mM KBrb
2 × 5 minutes, 22 °C 60 minutes, 4 °C 2 × 5 minutes
GABA GABAA Muscimol
[7.7nM]
GABA
[10 μM]
50 mM Tris-citrate (pH 7.0) 3 × 5 minutes, 4 °C 40 minutes, 4 °C 3 × 3 seconds
  GABAB CGP 54626
[2 nM]
CGP 55845
[100 μM]
50 mM Tris-HCl (pH 7.2)
+ 2.5 mM CaCl2
3 × 5 minutes, 4 °C 60 minutes, 4 °C 3 × 2 seconds
  BZ Flumazenil
[1 nM]
Clonazepam
[2 μM]
170 mM Tris-HCl (pH 7.4) 15 minutes, 4 °C 60 minutes, 22 °C 2 × 1 seconds
  pBZ PK 11195
[0.1 nM]
PK 11195
[10 μM]
50 mM Tris-HCl (pH 7.4) 15 minutes, 22 °C 60 minutes, 22 °C 3 × 5 minutes
Acetylcholine M1 Pirenzepine
[1 nM]
Pirenzepine
[2 μM]
Krebs buffer (pH 7.4)
+ 4 mM KCl
+ 120 mM NaCl
15 minutes, 22 °C 60 minutes, 22 °C 3 × 4 minutes
  M2 Oxotremorine-M
[1.7 nM]
Carbachol
[10 μM]
20 mM HEPES-Tris (pH 7.5)
+ 10 mM MgCl2
+ 300 nM pirenzepine
20 minutes, 22 °C 60 minutes, 22 °C 2 × 2 minutes
  M3 4-DAMP
[1 nM]
Atropine sulfate
[10 μM]
50 mM Tris-HCl (pH 7.4)
+ 0.1 mM PMSF
+ 1 mM EDTA
15 minutes, 22 °C 45 minutes, 22 °C 2 × 5 minutes
  nACh Epibatidine
[0.5 nM]
Nicotine
[100 μM]
1 mM HEPES (pH 7.5)
+ 120 mM NaCl
+ 5.4 mM KCl
+ 0.8 mM MgCl2
+ 1.8 mM CaCl2
20 minutes, 22 °C 90 minutes, 22 °C 5 minutes
Noradrenaline α1 Prazosin
[0.2 nM]
Phentolamine mesylate
[10 μM]
50 mM Na+/K+-phosphate buffer (pH 7.4) 15 minutes, 22 °C 60 minutes, 22 °C 2 × 5 minutes
  α2 UK 14,304
[0.64 nM]
Phentolamine mesylate
[10 μM]
50 mM Tris-HCl (pH 7.7)
+ 100 μM MnCl2
15 minutes, 22 °C 90 minutes, 22 °C 5 minutes
Serotonin 5-HT1A 8-OH-DPAT
[1 nM]
5-Hydroxytryptamine
[1 μM]
170 mM Tris-HCl (pH 7.4)
+ 4 mM CaCl2 b
+ 0.01% ascorbateb
30 minutes, 22 °C 60 minutes, 22 °C 5 minutes
  5-HT2 Ketanserin
[1.14 nM]
Mianserin
[10 μM]
170 mM Tris-HCl (pH 7.7) 30 minutes, 22 °C 120 minutes, 22 °C 2 × 10 minutes
Dopamine D1 SCH 23390
[1.67 nM]
SKF 83566
[1 μM]
50 mM Tris-HCl (pH 7.4)
+ 120 mM NaCl
+ 5 mM KCl
+ 2 mM CaCl2
+ 1 mM MgCl2
20 minutes, 22 °C 90 minutes, 22 °C 2 × 10 minutes
  D2 Raclopride
[0.55 nM]
Butaclamol
[1 μM]
50 mM Tris-HCl (pH 7.4)
+ 0.1% ascorbate
+ 150 mM NaCl
20 minutes, 22 °C 45 minutes, 22 °C 6 × 1 minutes
Adenosine A1 DPCPX
[1 nM]
R-PIA
[100 μM]
170 mM Tris-HCl (pH 7.4)
+ 2 U/L adenosine deaminase
+ 100 μM Gpp(NH)pb
15 minutes, 4 °C 120 minutes, 22 °C 2 × 5 minutes
  A2A ZM 241385
[0.42 nM]
2-Chloroadenosine
[20 μM]
170 mM Tris-HCl (pH 7,4)
+ 2 U/L adenosine deaminase
+ 10 mM MgCl2
2 × 10 minutes, 22 °C 120 minutes, 22 °C 2 × 5 minutes
  1. Abbreviations: A 1 Adenosine receptor type 1, A 2A Adenosine receptor type 2A, α 1 Adrenoceptor type 1, α 2 Adrenoceptor type 2, AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, BZ γ-Aminobutyric acid type A-associated benzodiazepine-binding site, D1 Dopamine receptor type 1, D2 Dopamine receptor type 2, 4-DAMP 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide, EDTA Ethylenediaminetetraacetic acid, GABA A γ-Aminobutyric acid receptor type A, GABA B γ-Aminobutyric acid receptor type B, Gpp(NH)p 5′-guanylimidodiphosphate, 5-HT 1A Serotonin receptor type 1A, 5-HT 2 Serotonin receptor type 2, KBr Potassium bromide, KSCN Potassium thiocyanate, M 1, M 2 , and M 3 Muscarinic cholinergic receptor types 1, 2, and 3, mGlu2/3 Metabotropic glutamate receptor type 2/3, MK-801 Dizocilpine, nACh Nicotinic cholinergic receptor of the α42 type, NMDA N-methyl-d-aspartate, 8-OH-DPAT 8-Hydroxy-2-(dipropylamino)tetralin, pBZ Peripheral benzodiazepine receptor, PMSF Phenylmethylsulfonyl fluoride, R-PIA N 6-R-phenylisopropyladenosine, SYM 2081 (2S,4R)-4-Methylglutamic acid
  2. a Final rinsing was carried out in incubation buffer at 4 °C, followed by one to three dips in distilled water at room temperature, unless otherwise specified
  3. b Substance included only in the main incubation buffer solution